JP2020060392A - Pre-treatment kit, medicinal toxicant screening pre-treatment kit, analysis sample preparation method, and analysis method - Google Patents

Abstract

To more rapidly prepare analysis samples for use in chromatography, or the like.SOLUTION: The pre-treatment kit for pre-treating samples to be used for at least one of chromatography and mass spectrometry, is provided with containers in each of which a compound for salting a solution containing the sample is arranged and the capacity of the container is less than 10 mL.SELECTED DRAWING: Figure 1

Description

  TECHNICAL FIELD The present invention relates to a pretreatment kit, a pretreatment kit for drug / toxic substance screening, a method for preparing an analytical sample, and an analytical method.

  Substances such as pesticide residues in foods and drug poisons in biological samples are detected by chromatography, mass spectrometry and the like. As a pretreatment for extracting these substances from a sample, the QuEChERS method, an improved method thereof, and the like are performed. In these methods, an organic solvent and a salt are added to a sample, and a contaminant component contained in the sample is removed by salting out (see Non-Patent Document 1).

Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ. "Fast and easy multiresidue method employing acetonitrile extraction / partitioning and" dispersive solid-phase extraction "for the determination of pesticide residues in produce" Journal of AOAC International, (USA), AOAC International, March 2003, Volume 86, Issue 2, pp.412-431

  The kit and the like used for the QuEChERS method and its improved method are premised on the presence of at least 500 μL of sample, and efficient analysis may not be possible if the number of obtained samples is small.

A pretreatment kit according to a preferred embodiment of the present invention is a pretreatment kit for a sample to be subjected to at least one of chromatography and mass spectrometry, in which a compound for salting out a solution containing the sample is arranged. A container is provided, and the capacity of the container is less than 10 mL.
In a more preferred embodiment, the volume of the container is 200 μL or more and less than 5 mL.
In a further preferred embodiment, a plurality of said containers are provided.
In a more preferred embodiment, the compound comprises a salt.
In a more preferred embodiment, the sample comprises a protein and the compound is for removing the protein by the salting out.
In a more preferred embodiment, said compound is at least one molecule selected from the group consisting of magnesium sulfate, sodium chloride, trisodium citrate, disodium hydrogen citrate and sodium acetate.
The pretreatment kit for drug toxicity screening according to a preferred embodiment of the present invention includes the pretreatment kit described above.
A method for preparing an analytical sample according to a preferred embodiment of the present invention is a method for preparing an analytical sample to be subjected to at least one of chromatography and mass spectrometry, in which a compound for salting out a solution containing the sample is arranged. Preparing a container having a volume of less than 10 mL, adding at least an organic solvent and a sample to the container, mixing them, and centrifuging the solution obtained by the mixing.
An analysis method according to a preferred embodiment of the present invention comprises preparing an analysis sample by the above-described method for preparing an analysis sample, and analyzing the analysis sample.
In a further preferred embodiment, the analysis is gas chromatography, liquid chromatography, mass spectrometry, gas chromatography / mass spectrometry, or liquid chromatography / mass spectrometry.

  According to the present invention, pretreatment such as the QuEChERS method utilizing salting out and its improved method can be efficiently performed even when the amount of the sample is small.

FIG. 1 is a conceptual diagram showing a pretreatment kit of one embodiment. FIG. 2 is a conceptual diagram for explaining a method for preparing an analytical sample according to one embodiment. FIG. 3 is a table showing the recovery rate of each benzodiazepine-based compound when a sample was stirred with or without beads in the analysis of Examples.

  Embodiments for carrying out the present invention will be described below with reference to the drawings.

-First Embodiment-
FIG. 1 is a conceptual diagram showing a pretreatment kit of this embodiment. The pretreatment kit 1 includes a plurality of containers (hereinafter referred to as pretreatment containers) 10 in which compounds 11 for salting out a solution containing a sample are stored. The pretreatment container 10 includes a pretreatment container body 12 and a lid portion 13.
The pretreatment kit 1 may include other articles used in pretreatment.

  The pretreatment kit 1 is a kit for performing pretreatment for analysis using at least one of chromatography and mass spectrometry. This analysis includes gas chromatography, liquid chromatography, mass spectrometry using MALDI or PESI, gas chromatography / mass spectrometry (hereinafter referred to as GC / MS) and liquid chromatography / mass spectrometry (hereinafter referred to as LC / MS). It is preferable for analyzing the liquid sample obtained by the pretreatment. Here, MALDI is an abbreviation for matrix-assisted laser desorption ionization, and PESI is an abbreviation for probe electrospray ionization. It is more preferable to use the pretreatment kit 1 for pretreatment of GC / MS and LC / MS which can detect each component of the sample with high precision by separating a plurality of times.

  Suitably, the pretreatment kit 1 is a kit for performing pretreatment for analysis of a biological sample derived from an organism, a reconstitution system or the like. This biological sample preferably contains a protein, and the pretreatment kit 1 is preferably a kit for deproteinizing the biological sample by salting out. The pretreatment kit 1 is more preferably a kit for performing the QuEChERS method or its improved method. It is more preferable that the pretreatment kit 1 is a pretreatment kit for toxic substance screening used for pretreatment for screening of toxic substances in forensic medicine or investigations and toxic substances such as pesticide residues in foods.

  The volume of the pretreatment container 10 is smaller than 10 mL. The volume of the pretreatment container 10 is preferably smaller than 5 mL, more preferably smaller than 3 mL, and further preferably smaller than 2.5 mL. The smaller the volume of the pretreatment container 10 is, the more suitable the container is for pretreatment when an analysis is performed with a small amount of sample, and since it is not unnecessarily large, the space occupied during storage can be saved. .

Furthermore, the inventor has found that the smaller the volume of the pretreatment container 10, the higher the effect of stirring. Therefore, the pretreatment kit 1 does not need to include beads. Thereby, the pretreatment kit 1 can be easily manufactured and the manufacturing cost can be reduced.
Since there is a possibility that the sample can be mixed more reliably than when beads are not arranged, beads such as stainless beads may be arranged in the pretreatment container 10.

  If the volume of the pretreatment container 10 is too small, the amount of the solution required for analysis cannot be stored or handling becomes difficult. Therefore, the volume of the pretreatment container 10 is preferably 200 μL or more, more preferably 500 μL or more.

  The shape of the pretreatment container 10 is not particularly limited, and the lid portion 13 may be integrally formed with the pretreatment container body 12, or the lid portion 13 and the pretreatment container body 12 are separated. It may be formed as a plurality of parts. Further, the pretreatment container 10 may be formed integrally with another pretreatment container 10, and for example, a pretreatment container body is provided for each predetermined number of pretreatment containers 10 such as an 8-tube. 12 or the lid portion 13 may be integrally formed.

  Although 24 pretreatment containers 10 are shown in FIG. 1, the number of pretreatment containers 10 included in the pretreatment kit 1 is not particularly limited. The number is 1 or more, preferably 2 or more, more preferably 5 or more, even more preferably 10 or more, and further preferably 20 or more. As the number of the pretreatment containers 10 included in the pretreatment kit 1 is larger, the work for purchasing the pretreatment container 10 containing the compound 11 and replenishing it can be reduced.

  If the number of the pretreatment containers 10 included in the pretreatment kit 1 is too large, the storage period becomes long and the activity of the compound 11 is decreased, or it is necessary to make a storage space wide so that other articles can be stored. Since such a problem arises that the space to be filled becomes narrower, the number can be appropriately set to 1000 or less or 500 or less.

  The compound 11 placed in the pretreatment container 10 is a compound for salting out the solution containing the sample. Compound 11 causes a salting-out when the solution is added to the pretreatment container 10 to obtain a salt solution having a sufficient concentration to remove impurities such that the analysis described below can be performed with desired accuracy. There is no particular limitation as long as it can be used. Compound 11 preferably comprises a salt.

  Suitably, compound 11 comprises at least one of anhydrous magnesium sulfate, sodium chloride, trisodium citrate, disodium hydrogen citrate and sodium acetate. Compound 11 preferably comprises at least one buffering agent selected from the group consisting of trisodium citrate, disodium hydrogen citrate and sodium acetate. The buffering agent can suppress the change in pH of the solution containing the sample and stabilize the component to be analyzed.

  The compound 11 is preferably stored in a solid state in each pretreatment container 10 for quality control, but it may be dissolved in a solvent and stored.

  In the pretreatment kit 1, since the compound 11 is subdivided and arranged in the pretreatment container 10, it is possible to perform the pretreatment quickly without the need to weigh the compound 11 and place it in the container. In particular, when the sample is small, the amount of the required compound 11 is small and the weighing may be difficult. However, even in such a case, the pretreatment can be performed accurately by using the pretreatment kit 1.

  The method for producing the pretreatment container 10 containing the compound 11 is not particularly limited. The compound 11 in a solid state may be weighed and placed in each pretreatment container 10, or a solution containing the compound 11 at a predetermined concentration may be prepared using a suitable solvent, and then a pipette, a dispensing device, or the like. Alternatively, a predetermined amount of the solution may be dispensed into each pretreatment container 10 and appropriately dried.

(Method for preparing sample for analysis)
FIG. 2 is a conceptual diagram showing a method for preparing an analytical sample using the pretreatment kit 1. In FIG. 2, the component of the sample S to be analyzed has a higher affinity for the organic solvent than the aqueous solvent, and the case of extracting the component into the organic solvent will be described as an example.

  In step S1001, the pretreatment container 10 in which the compound 11 is arranged and the sample S are prepared. The sample S is placed in the pretreatment container 10. In FIG. 2, the sample S is dispersed in the solution and the pipette P1 dispenses the solution into the pretreatment container 10, but the dispensing method is not particularly limited. Further, the sample S may be placed in the pretreatment container 10 as it is as a solid. When step S1001 ends, step S1002 starts (arrow A1).

In step S1002, an organic solvent is added to the pretreatment container 10 containing the sample S and the compound 11. The substance that constitutes the organic solvent is not particularly limited, and for example, acetonitrile or the like can be used. In FIG. 2, the organic solvent is dispensed by the pipette P2, but the method of dispensing the organic solvent is not particularly limited. When the amount of water contained in the solution S1 containing the sample S is not sufficient, it is preferable to add water appropriately. Furthermore, a predetermined amount of an internal standard (not shown) is added to the solution S1 containing the sample S. When step S1002 ends, step S1003 starts (arrow A2).
The order of adding the sample S, the organic solvent, water, and the internal standard to the pretreatment container 10 is not particularly limited.

  In step S1003, the solution S1 containing the sample S is stirred. The sample S, the compound 11, water, the organic solvent, and the internal standard are mixed, and the solution S1 containing the sample S is subjected to salting out. Contaminant components such as proteins originally contained in the sample S are precipitated by salting out. The stirring method is not particularly limited, and it can be performed using a vortex mixer or the like. The solution after stirring is subjected to centrifugation by a centrifuge. The method of centrifugation is not particularly limited, and the centrifugation can be performed, for example, at 10,000 rpm for 10 minutes. When step S1003 ends, step S1004 starts (arrow A3).

In step S1004, the supernatant Sn of centrifugation is extracted. After the centrifugation in step S1003, the analysis target component of the sample S is extracted into the organic solvent layer (supernatant Sn), and the contaminant components and the like are present in the aqueous layer or as a precipitate. The method for extracting the supernatant Sn is not particularly limited and can be performed using a pipette or the like. When step S1004 ends, step S1005 starts (arrow A4).
The solid phase extraction may be performed by adding at least one of primary and secondary amines (PSA), graphite carbon, magnesium sulfate and C18 to the solution obtained by the centrifugation in step S1003. PSA is effective in removing sugars, fatty acids, organic acids and pigments, graphite carbon and C18 are effective in removing non-polar impurities, and magnesium sulfate is effective in removing water.

  In step S1005, the supernatant Sn is prepared as a sample for analysis and provided for analysis. The supernatant Sn is subjected to at least one of chromatography and mass spectrometry. The supernatant Sn is introduced into the analyzer as a sample for analysis, and when performing mass spectrometry by MALDI, a matrix is added to the supernatant Sn to prepare a crystalline sample for analysis, and the sample is provided for ionization.

  The method for preparing an analytical sample according to the present embodiment is a method for preparing an analytical sample to be subjected to at least one of chromatography and mass spectrometry, in which a compound 11 for salting out a solution S1 containing a sample S is arranged. And preparing a pretreatment container 10 having a volume smaller than 10 mL, adding an aqueous solvent, an organic solvent or a mixed solution thereof, and a sample S to the pretreatment container 10 and mixing them. Centrifuging the solution obtained by. As a result, the advantages of the pretreatment kit 1 can be taken advantage of, and pretreatment such as the QuEChERS method utilizing salting out and its improved method can be efficiently performed even when a small amount of sample is used.

  The analysis method according to the present embodiment includes preparing an analysis sample by the method for preparing an analysis sample according to the present embodiment, and analyzing the analysis sample. As a result, the advantages of the pretreatment kit 1 can be taken advantage of, and the analysis involving pretreatment such as the QuEChERS method utilizing salting out and its improved method can be efficiently performed even with a small amount of sample.

  The present invention is not limited to the contents of the above embodiment. Other aspects that are conceivable within the scope of the technical idea of the present invention are also included within the scope of the present invention.

  The following is an example in which the influence of the presence or absence of beads on the sample recovery rate was examined, but the present invention is not limited to the specific numerical values and analysis conditions of the following examples.

In the pretreatment, the recovery rate was calculated based on the peak area of the mass spectrum in the case where 34 kinds of benzodiazepine compounds were added before mixing for salting out and the case where they were added after mixing. The recovery rates were compared between the case where beads (3 pieces) having a diameter of 3 mm were arranged in the pretreatment container and the case where they were not arranged.
<1. Pretreatment>
100 mg salt consisting of magnesium sulfate and sodium acetate was placed in a 2.0 mL tube. A tube containing stainless beads and a tube not containing stainless beads were prepared. To these tubes, 270 μL of acetonitrile, 30 μL of a mixed solution containing an internal standard, and 200 μL of distilled water were added, and mixed with a vortex mixer. To the solution obtained by mixing, 2 μL of 10 μg / μL diazepam-d5 was added, and 100 μL of whole blood was added and mixed with a vortex mixer. The solution obtained by mixing was centrifuged at 10,000 rpm for 10 minutes.

<2. LC / MS>
The supernatant obtained by centrifugation was subjected to liquid chromatography / tandem mass spectrometry under the following conditions.
Liquid Chromatography Conditions Analytical column: Kinetex XB-C18 (Phenomenex) (internal diameter 2.1 mm, length 100 mm, particle size 2.6 μm), guard column: Security Guard Ultra (Phenomenex) (internal diameter 2.1 mm), injection amount 1 μL, column temperature: 40 ° C., mobile phase: (A) 10 mM ammonium formate + 0.1% formic acid aqueous solution; (B) 10 mM ammonium formate + 0.1% formic acid methanol solution, flow rate: 0.3 mL / min.

Conditions for mass spectrometry Temperature: Desolvation line (DL) temperature: 250 ° C, heat block temperature: 400 ° C, interface temperature: 300 ° C. Gas flow rate: nebulizer gas flow rate: 2 L / min, drying gas flow rate: 10 L / min, heating gas flow rate: 10 L / min.

  FIG. 3 is a table showing the results of the analysis. No significant difference in recovery was observed with and without the addition of stainless beads.

1 ... Pretreatment kit, 10 ... Pretreatment container, 11 ... Compound, S ... Sample, S1 ... Solution containing sample, Sn ... Supernatant.

Claims (10)

A kit for pretreatment of a sample to be subjected to at least one of chromatography and mass spectrometry, comprising:
A container for arranging a compound for salting out a solution containing the sample,
A pretreatment kit having a capacity of less than 10 mL. The pretreatment kit according to claim 1,
A pretreatment kit having a capacity of 200 μL or more and less than 5 mL. The pretreatment kit according to claim 1 or 2,
A pretreatment kit comprising a plurality of the containers. The pretreatment kit according to any one of claims 1 to 3,
The compound is a pretreatment kit containing a salt. The pretreatment kit according to any one of claims 1 to 4,
The sample comprises protein,
The compound is a pretreatment kit for removing the protein by the salting out. The pretreatment kit according to any one of claims 1 to 5,
The pretreatment kit, wherein the compound is at least one molecule selected from the group consisting of magnesium sulfate, sodium chloride, trisodium citrate, disodium hydrogen citrate and sodium acetate.   A pretreatment kit for drug poisoning screening, comprising the pretreatment kit according to any one of claims 1 to 6. A method for preparing an analytical sample for use in at least one of chromatography and mass spectrometry, comprising:
Preparing a container for arranging a compound for salting out a solution containing a sample, the volume of which is smaller than 10 mL;
Adding at least an organic solvent and a sample to the container and mixing;
A method for preparing an analytical sample, which comprises centrifuging the solution obtained by the mixing. Preparing an analytical sample by the method for preparing an analytical sample according to claim 8;
Performing an analysis of the analytical sample,
An analysis method comprising. The analysis method according to claim 9,
The analysis method is gas chromatography, liquid chromatography, mass spectrometry, gas chromatography / mass spectrometry, or liquid chromatography / mass spectrometry.

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